1. Field of the Invention
The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between the ligand, or analyte under determination, and a binding partner therefor. Where one of the ligand and its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In conventional specific binding assay techniques, a sample of the liquid medium to be assayed is combined with reagent means of various compositions. Such compositions include a labeled conjugate comprising a binding component incorporated with a label. The binding component in the labeled conjugate participates with other constituents, if any, of the reagent means and the ligand in or ligand binding capacity of the medium under assay to form a binding reaction system producing two species or forms of the labeled conjugate, a bound-species and a free-species. In the bound-species, the binding component, e.g., a hapten or antigen, in the labeled conjugate is bound by a corresponding binding partner, e.g., an antibody, whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand or ligand binding capacity to be detected in the test sample.
Where the labeled conjugate in the bound-species is essentially indistinguishable in the presence of the labeled conjugate in the free-species by the means used to monitor the label, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to in the art as "heterogeneous". Where the bound-species and free-species forms of the labeled conjugate can be distinguished in the presence of each other, a "homogeneous" format can be followed and the separation step avoided.
This invention relates to assay methods and reagent means of the homogeneous specific binding type of determining qualitatively or quantitatively a ligand in a liquid medium. In particular, the invention relates to an improved homogeneous specific binding assay employing a novel intramolecularly modulated photogenic enzyme substrate as the label component in the labeled conjugate.
2. Description of the Prior Art
The first discovered type of highly sensitive specific binding assay was the radioimmunoassay which employs a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials and the necessity of a separation step, homogeneous assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme activators and inhibitors, cycling reactants, organic and inorganic catalysts, prosthetic groups, chemiluminescent reactants, and fluorescent molecules.
A homogeneous specific binding assay employing an enzyme-cleavable substrate label is described by Burd et al, Anal. Biochem. 77:56(1977) and in German OLS No. 2,618,511 and British Pat. No. 1,552,607 corresponding to U.S. patent application Ser. No. 667,996, filed Mar. 18, 1976, assigned to the present assignee. In exemplified embodiments there is disclosed the use of fluorogenic-labeled conjugates comprising a fluorescer, e.g., umbelliferone or fluorescein, coupled through an enzyme-cleavable bond, e.g., an ester bond, to an appropriate binding component. Cleavage of the linking bond releases the free fluorescer. In forming the labeled conjugate, the fluorescer is coupled at a site which alters its fluoroescence properties. e.g., the fluorescer moiety in the labeled conjugate is essentially nonfluorescent upon exposure to light at the wavelength which causes the enzymatically released fluorescer to fluoresce. In this assay, the rate of fluorescence production, which follows the rate of release of the fluorescer, is a function of the concentration of ligand in the medium under assay.
An improved homogeneous enzyme substrate-labeled specific binding assay is described by Burd et al, Clin. Chem. 23:1402(1977) and in U.S. Pat. No. 4,226,978, assigned to the present assignee. The improvement comprises employing as the label component in the labeled conjugate, a residue of the formula: EQU G--D--R
wherein G is a glycone, e.g., .beta.-galactosyl, D is a dye indicator moiety, preferably a fluorescer, e.g., umbelliferone, and R is the linkage to the appropriate binding component. In the presence of a suitable glycosidase enzyme, e.g., .beta.-galactosidase, the glycone moiety is cleaved to release a dye-(binding component) product which is distinct in its fluorescence properties from the labeled conjugate. As in the previously described system, the dye indicator moiety, e.g., fluorescer, is coupled at a site which alters its fluorescence properties. Also, since the fluorescent product of the enzymatic cleavage is a conjugate of the fluorescer and the binding component, performance of the assay is dependent on there being no substantial modulation of the fluorescer by the binding component. Any substantial native modulating character of the binding component could affect the function of the assay.
It is an object of the present invention to provide a homogeneous enzyme substrate-labeled specific binding assay based on a fluorescence detection system, or similar photogenic system (characterized by the production or emission of light), wherein the fluorescer, or photophore, need not be coupled at a critical site in constructing the labeled conjugate, and wherein any native modulating character of the conjugated binding component relative to the photogenicity of the photophore does not affect the function of the assay system.
Of ancillary relevance to the present invention are Weber, Flavins and Flavoproteins, ed. Slater, Elsevier (Amsterdam 1966) pp. 15-21; and Forster, Fluoreszenz organischer Verbindungen, Vandenhoeck u. Ruprecht (Gottingen 1951) relating to the phenomena of intramolecular collision and energy transfer, and Stryer, Ann. Rev. Biochem. 47:819 (1978); Yaron et al, Anal. Biochem. 95:228(1979); Ullman et al, J. Biol. Chem. 251:4172(1976); U.S. Pat. No. 3,996,345; British Published Patent Application No. 2,018,424A; and Japanese Laid Open Patent Application No. 53,142,522, relating to the application of the intramolecular energy transfer phenomenon to analytical methods, including enzyme assays and immunoassays.